PROJECT SUMMARY Chronic exposure to nicotine in tobacco products results in numerous health consequences (lung cancer, emphysema, hypertension, etc.) and accounts for over 6 million deaths per year. Relapse rates are high among those who attempt to quit smoking, and pharmacotherapies that seek to foster smoking cessation have moderate effectiveness. Thus, there is a significant unmet need for more effective strategies to treat nicotine dependence. Development of such strategies requires a more detailed understanding of the biological mechanisms leading to nicotine addiction. An essential goal related to mechanistic studies on nAChRs is gaining a better understanding of the location and activity of nAChRs in discrete sites within individual nerve cells. Although some basic research studies have begun to describe nAChR subcellular distribution, there is currently no plausible way to functionally interrogate nAChRs at the subcellular level. This means we are currently unable to determine whether the important nicotine-mediated functional alterations in nAChRs occur in dendrites, axons, presynaptic terminals, or in neuronal somata. Answering this key question is absolutely required for the field to fully understand the molecular and cellular basis for nicotine dependence. Here, we propose a R21/R33 phased innovation project that directly addresses these critical gaps in our ability to study native nAChRs. The R21 phase will develop and characterize a photoactivatable nicotine (PA-Nic) compound for use in nicotine ?uncaging? experiments. R21 Aim 1 focusses on identification of 1 or more suitable compounds using ultraviolet/visible light optical methods, while Aim 2 will characterize promising compounds using 2-photon uncaging techniques, which offer enhanced spatial resolution. In the R33 phase, we propose to employ these innovative compounds and optical methods in discovery experiments designed to uncover new details about nAChR function. R33 Aim 1 will involve functional mapping of nAChRs on key cell types involved in nicotine dependence. R33 Aim 2 will probe how these receptors enable nicotine to participate in circuit-level modulation of neurotransmission in brain's reward system. This project represents a substantial technical advance for the cholinergic biology field, as it will not only produce new tools for widespread use, but will utilize those tools to uncover new mechanistic details about nicotine dependence.